Methods and compositions for prevention and treatmentof cardiac hypertrophy

ABSTRACT

Methods are provided of treating cardiac hypertrophy in a mammalian subject comprising administering to the subject an anti-hypertrophic effective amount of an ion channel TR-PV1 inhibitor. The methods include treatment of a symptom of cardiac hypertrophy in the subject comprises cardiac remodeling, cardiac fibrosis, apoptosis, hypertension, or heart failure

PRIORITY CLAIM

Priority is claimed pursuant to 35 USC 119(e) of U.S. provisionalapplication Ser. No. 61/409,781, filed Nov. 3, 2010, the disclosure ofwhich is incorporated by reference herein in its entirety for allpurposes.

U.S. GOVERNMENT RIGHTS

This invention was made with U.S. Government support under grant NCRRU54RR026136; NAIPI P20RR016467; NIMHD P20MD006084; NCRR 5P20RR016453_from the National Institutes Health. The U.S. Government may havecertain license rights in this invention.

FIELD OF THE INVENTION

The present invention relates to the treatment and prevention cardiachypertrophy. More specifically, the invention relates to methods andcompositions for preventing or treating cardiac hypertrophy, cardiacremodeling, fibrosis, hypertension, and heart failure in mammals,including humans, through inhibition of the ion channel TRPV1.

BACKGROUND

Myocardial hypertrophy is the fundamental response of the heart to achronically increased workload, which can result from conditions such ashypertension or valve disorders. The progression of myocardialhypertrophy represents a principal risk factor for the development ofheart failure and subsequent cardiac death.

The focus of this invention is on combating hypertrophy, apoptosisfibrosis, and heart failure, focuses on regulation of TRPV1 (transientreceptor potential cation channel, subfamily V, member 1), a complex andremarkable receptor/channel. TRPV1 is typically classified as anocioceptive receptor. Published data indicate that the open probabilityof TRPV1 is controlled by the endocannabinoid anandamide, its endogenousligand, and pathways modulating anandamide levels also influence TRPV1activation. The etiology of hypertrophic regulation by TRPV1 (transientreceptor potential cation channel, subfamily V, member 1) is unknown.There is only a general understanding of how TRPV1 is regulated, and ofthe identity of several cell and tissue types in which TRPV1 resides.

TRPV1 has been studied in peripheral sensory neurons as a pain receptor;however TRPV1 is expressed in numerous tissues and cell types includingthose of the cardiovascular system. TRPV1 expression is upregulated inthe hypertrophic heart, and the channel is positioned to receivestimulatory signals in the hypertrophic heart. TRVP1 is a sixtrans-membrane tetrameric nonselective cation channel, typicallyassociated with peripheral sensory neurons involved in nociception.Exogenous activators of TRPV1 include temperature of greater than 43° C.and capsaicin. Endogenously, TRPV1 is activated and potentiated by theendocannabinoids, anandamide and N-arachidonoyl-dopamine, low pH, andphosphorylation by protein kinase C (PKC) and cyclic AMP-dependentprotein kinase (PKA). The nociceptive involvement of TRPV1 activation inperipheral sensory neurons has prompted substantial study of TRPV1 as atarget for inhibition. Consequently a plethora of effective TRPV1antagonists has been produced and demonstrated to be effectiveanalgesics in the management of inflammatory pain and hyperalgesia.

In addition to the peripheral sensory neurons, TRPV1 is also found inother excitable and non-excitable tissues, including those of the heartand circulatory system. For example, cardiomyocytes, cardiac bloodvessels, perivascular nerves, pulmonary artery smooth muscle cells, andcoronary endothelial cells, skeletal muscle, mast cells, and dendriticcells express TRPV1.

Although TRPV1 inhibition has not been studied in the context of cardiachypertrophy, TRPV1 activation has been implicated in protection frommyocardial ischemia reperfusion injury. In addition, the channel'sendogenous ligand, anandamide, has been implicated in multiple cardiacdiseases such as cardiotoxicity and hypertension.

SUMMARY OF EXEMPLARY EMBODIMENTS OF THE INVENTION

The invention provides methods of treating cardiac hypertrophy in amammalian subject comprising administering to the subject ananti-hypertrophic effective amount of an ion channel TRPV1 inhibitor. Insome embodiments, the invention provides methods of treatment where asymptom of cardiac hypertrophy in the subject comprises cardiacremodeling, cardiac fibrosis, apoptosis, hypertension, or heart failure.

The invention further provides methods prophylactic treatment forcardiac hypertrophy in a mammalian subject comprising administering tothe subject an anti-hypertrophic effective amount of an ion channelTRPV1 inhibitor.

The invention also provides pharmaceutical compositions comprising apharmaceutically effective amount of an ion channel TRPV1 inhibitor ormixtures of such inhibitors useful for the methods of the invention.

The invention also provides pharmaceutical compositions comprising apharmaceutically effective amount of the ion channel TRPV1 inhibitor(N-(4-t-butyl-phenyl)-4-(3-chloropyridin-2-yl)-tetrahydropyrazine-1(2H)-carboxamide or mixtures of that inhibitor with other ion channelTRPV1 inhibitors useful for the methods of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D are graphs of results of data obtained by the proceduresdescribed in Example 1.

FIG. 2 is a graph of results of data obtained by the picosirius stainingprocedure described in Example 2.

FIGS. 3A-3B are graph of results of data obtained by the TGF-beta RNAexpression and ANP expression procedures described in Example 2.

FIGS. 4A-4B are graphs of results of data obtained by the proceduresdescribed in Example 3.

FIGS. 5A-5F are graphs of test results obtained by procedures describedin Example 4 pertaining to gravimetric, structural, and functionalanalysis of the heart during and after applied pressure overload cardiachypertrophy.

FIGS. 6A-6D are graphs of test results on measurement of cardiomycytecross sectional area, and expression levels of ANP and TGFβ byprocedures described in Example 4.

FIGS. 7A-7E are graphs of test results on measurement of fibrosis,tissue remodeling, and inflammatory markers by procedures described inExample 4.

FIGS. 8A-8E are graphs of test results on measurement of heart mass,structure and function during pressure overload cardiac hypertrophy byprocedures described in Example 5.

FIGS. 9A-9B are graphs of measurements from histological analysis ofmice treated with the TRPV1 antagonist BCTC according to proceduresdescribed in Example 6.

DETAILED DESCRIPTION EXEMPLARY EMBODIMENTS OF THE INVENTION

Cardiac hypertrophy is classically considered to be an adaptive andcompensatory response that increases the work output of cardiomyocytesand thus maintains cardiac function despite increased load. In mice,cardiac hypertrophy is typically modeled using transverse aorticconstriction (TAC) to induce acute pressure overload. The increasedresistance created by aortic constriction initially compromises leftventricular (LV) function; the subsequent development of LV hypertrophybegins to restore systolic function in the two weeks following TAC.Concentric LV hypertrophy continues during weeks two to eleven post-TAC,potentially doubling the LV mass compared to controls. A decline in LVfunction accompanies LV chamber dilation and myocardial fibrosis, andaround half of TAC treated mice develop pulmonary congestion by weekeleven. Thus, TAC is an effective stimulus for rapidly producing cardiachypertrophy in an experimental setting. The TAC model providestremendous utility for identifying important therapeutic targets inheart disease and exploring the effects of molecular or pharmacologicalinhibitors.

This invention shows that TRPV1 function is a new target for protectivetherapy in cardiac hypertrophy, fibrosis and heart failure usingTRPV1-directed therapeutics, this invention has the potential to shiftclinical treatment paradigms for cardiac hypertrophy and heart failureby repurposing existing drugs.

As shown below in the examples the loss of TRPV1 function in mice altersthe responses of the heart to TAC-induced pressure overload. TRPV1contributes to cardiac hypertrophy, fibrosis, apoptosis, and loss ofcontractile function in response to pressure overload. TRPV1 antagonistspreviously known as anti-hyperalgesics are unexpectedly provided by themethods of the present invention as anti-hypertrophic agents.

As shown in the examples the knockout of Trpv1 significantly suppressesthe ventricular enlargement, apoptosis, tissue remodeling and fibrosisassociated with modeled pressure overload cardiac hypertrophy. Thisphenotype mirrors some of the most desirable effects foranti-hypertrophic treatments. By use of a transverse aortic constrictionto model pressure overload cardiac hypertrophy mice lacking functionalTRPV1, compared to wild type, have improved heart function, and reducedhypertrophic, fibrotic and apoptotic markers. TRPV1 plays a role in theprogression of cardiac hypertrophy, and presents a therapeutic targetfor the treatment of cardiac hypertrophy and subsequent disease statesincluding arrhythmias, kidney dysfunction and heart failure; treatmentand alleviation of symptoms leading to cardiac hypertrophy and heartfailure such as high blood pressure, heart valve disease, weakness ofthe heart muscle (cardiomyopathy), abnormal heartbeat, anemia, thyroiddisorders, excessive drug use, muscular dystrophy and Fabry's disease,aortic valve stenosis, side effects of chemotherapy agents leading totoxic cardiomyopathy, obesity, diabetes, cigarette smoking, viralmyocarditis (an infection of the heart muscle), infiltrations of themuscle such as amyloidosis, HIV cardiomyopathy (caused by humanimmunodeficiency virus), connective tissue diseases such as systemiclupus erythematosus, abuse of drugs such as alcohol and cocaine, andside effects of arrhythmias or pharmaceutical drugs such aschemotherapeutic agents.

In addition to the compounds and compositions having activity herein,other compounds having the requisite activity may be identified by thefollowing test. Since cardiac hypertrophy is classically considered tobe an adaptive and compensatory response that increases the work outputof cardiomyocytes and thus maintains cardiac function despite increasedload, the following test will identify a compound as having the activityuseful in accordance with the invention. In mice, cardiac hypertrophy istypically modeled using transverse aortic constriction (TAC) to induceacute pressure overload. The increased resistance created by aorticconstriction initially compromises left ventricular (LV) function; thesubsequent development of LV hypertrophy begins to restore systolicfunction in the two weeks following TAC. Concentric LV hypertrophycontinues during weeks two to eleven post TAC, potentially doubling theLV mass compared to controls. A decline in LV function accompanies LVchamber dilation and myocardial fibrosis, and around half of TAC treatedmice develop pulmonary congestion by week eleven. Thus, TAC is aneffective stimulus for rapidly producing cardiac hypertrophy in anexperimental setting. Although there are differences between the TACmodel and clinical cardiac hypertrophy, this model mimics the acuteonset of hypertension rather than the gradual onset in clinical cases.However, the TAC model is useful for identifying important therapeutictargets in heart disease and exploring the effects of molecular orpharmacological inhibitors. (Lygate, 2006; Patten and Hall-Porter, 2009)

Test Model Generation of the Model in the Mouse Transverse AorticConstriction (TAC).

Transverse aortic constriction was performed as described by Rockman,producing left ventricular hypertrophy by constriction of the aorta(Rockman et al., 1994; Rockman et al., 1991). The left side of the chestwas depilated with Nair and a baseline 2-D echocardiogram was obtained.Mice were then deeply anesthetized with a mixture of ketamine andxylazine. The transverse aorta between the brachiocephalic and leftcarotid artery was banded using 6-0 silk ligature around the vessel anda 26G blunt needle, after which the needle was withdrawn. Sham surgerieswere identical apart from the constriction of the aorta.

Checking for Successful Banding Doppler Echocardiography.

Doppler echocardiography was performed one week post TAC to measure thelevel of constriction. Mice were anesthetized lightly with isofluorenegas and shaved. Doppler was performed using the Visualsonics Vevo 770system. In the parasternal short-axis view, the pulsed wave Dopplersample volume was placed in the transverse aorta just proximal anddistal to the site of banding. Peak velocity was traced using Vevo 770software, and the pressure gradient was calculated using the simplifiedBernoulli equation.

Following the Structural Changes in Heart Dimensions During theProgression of the Modeled Disease Transthoracic Echocardiography.

Baseline and post TAC transthoracic echocardiography were used to assesschanges in mouse heart dimensions and function. Briefly, after two daysof acclimatization and depilation, unanesthetized transthoracicechocardiography was performed using a 30-Mhz transducer (Vevo 770,VisualSonics). High quality two-dimensional images and M-mode images ofthe left ventricle were recorded. Measurements of left ventricularend-diastolic (LVIDd) and end-systolic (LVIDS) internal dimensions wereperformed by the leading edge to leading edge convention adopted by theAmerican Society of Echocardiography. The left ventricular ejectionfraction (% EF) was calculated as (LV Vol; d-LV Vol; s/LV Vol; d×100)(Visualsonics Inc.).

Testing the Degree of Cellular Hypertrophy, Fibrosis, and Apoptosis PostTreatment

Markers of hypertrophy, fibrosis, tissue remodeling, inflammation andapoptosis, are assessed by either Western Blot (WB) analysis, real-timePCR of extracted RNA (RT), or histological and immunohistologicalanalysis (H).

Hypertrophic Markers can Include:

ANP^((WB, RT)), BNP^((RT)), ACTA1^((RT)), α-MHC^((RT)), β-MHC^((RT)),MLC2A^((RT)), and Wheat-germ agglutinin^((H)) to generate cardiomyocytecross sectional area.

Tissue Remodeling Markers can Include:

Chymase CMA1^((WB, RT)), MMP-2^((RT)), MMP-9^((RT)), TGF-β^((RT)),Collagen III^((RT,H)), fibrinogen^((RT, H)), Fibroblast proliferationCD29^((H)).

Apoptosis Markers can Include:

Cleaved Caspase-3^((WB)).

Immunological, Inflammatory and Infiltration Markers can Include:

IL-6^((RT)), TNF-α^((RT)), NOS3^((RT)), CD68 (Macrophages)^((WB,RT,H)),histidine decarboxylase^((WB)) and Fc R1α^((WB)) (Mastcells)^((WB,RT,H)), CD4⁺/CD8⁺ T-cell markers^((WB,H)), NK cellCD161^((RT,WB,H))

Tissue Preparation for Histology.

Eight weeks post TAC, mice were euthanized by CO₂ asphyxiation, andhearts were collected for histological and molecular analysis. Forhistology, hearts were perfused with phosphate-buffered saline and 10%formalin in situ, collected immediately, and fixed overnight in 10%formalin at 4° C. Tissues were then cut in a sagittal orientation,embedded in paraffin, mounted on glass slides, and stored until use.Paraffin-embedded sections were stained for the following:

Collagen:

Collagen volume fraction was determined by analysis of picrosiriusstained sections. Sections cut to 5 μm thickness were deparaffinized,stained with Weigert's hematoxylin, then stained with picrosirius red(0.1% Sirius Red in picric acid). Sections were subsequently washed anddehydrated before image analysis.

Cardiomyocyte Cross Sectional Area:

Heart sections were deparaffinized and permeabilized, then stained withwheat germ-agglutinin conjugated to Alexa488 (WGA-Alexa488, Invitrogen,W11261) at a concentration of 50 μg/mL to identify sarcolemmal membranesand measure cardiomyocyte cross sectional area (described below).

Image Collection and Analysis.

Fluorescent and bright field images were collected on anepifluorescence-microscope (Axioscope, Zeiss). Fibrosis andcross-sectional cardiomyocyte area were quantified using ImageJ software(NIH). To quantify fibrosis, collagen fibers were highlighted, and thered-stained pixels were counted to determine the percentage of pixels ineach field that represented collagen fibers. Perivascular tissue wasexcluded from this calculation. Three heart sections from each animalwere imaged at five images per heart. Images were averaged for eachanimal and graphed in Prism GraphPad. Cardiomyocytes from WGA stainedsections were randomly selected in a blinded fashion then traced todetermine the cross sectional area of individual myocytes (n=100).

All images were captured and analyzed in a single-blind manner, exceptfor WGA staining, which was analyzed in a double-blind manner.

RT-PCR.

For RNA extraction, hearts were collected from mice and total RNA wasisolated from homogenized hearts with Trizol (Molecular Research Center,TR 118) and further purified with an RNA isolation kit (Mo BioLaboratories, Inc, 15000-250). Single-stranded cDNA was synthesized from1 ug of total RNA using a cDNA synthesis kit (Qiagen, 205113). The mRNAlevels of chymase (CMA1), atrial natriuretic peptide (ANP), TGF-,collagen III, matrix metalloproteinase (MMP) 2 and 9 and cyclophilin(CPN) were quantified by RT-PCR in triplicate with QuantiTect SYBR Green(Qiagen, 204245) in an Opticon device (MJ Research, Waltham, Mass.). Thefollowing primer pairs were used: ANP, 5′-AGA AAC CAG AGA GTG GGC AGAG-3′ (SEQ ID NO. 1) and 5′-CAA GAC GAG GAA GAA GCC CAG-3′ (SEQ ID NO.2); TGFβ, 5′-TGG AGC AAC ATG TGG AAC TC-3′ (SEQ ID NO. 3) and 5′-CAG CAGCCG GTT ACC AAG-3′ (SEQ ID NO. 4); MMP2,5′-TGG TGT GGC ACC ACC GAG GA-3′(SEQ ID NO. 5) and 5′-GCA TCG GGG GAG GGC CCA TA-3′ (SEQ ID NO. 6);MMP9,5′-CGG CAC GCC TTG GTG TAG CA-3′ (SEQ ID NO. 7) and 5′-TCG CGT CCACTC GGG TAG GG-3′ (SEQ ID NO. 8); Collagen III, 5′-GAC CGA TGG ATT CCAGTT CG-3′ (SEQ ID NO. 9) and 5′-TGT GAC TCG TGC AGC CAT CC-3′ (SEQ IDNO. 10); CMA1,5′-AGC TCA CTG TGC GGG AAG GTC T-3′ (SEQ ID NO. 11) and5′-CTC AGG GAC CAG GCA GGG CTT-3′ (SEQ ID NO. 12).

Western Blot Analysis.

Hearts were collected, and protein extracts were prepared fromhomogenized heart tissue using IGEPAL. Total protein concentrations weredetermined by the bicinchoninic acid (BCA) colorimetric assay.Absorbance was measured at 562 nm by spectrophotometer (Spectra Max340), and concentrations determined using a standard curve based onbovine serum albumin (BSA) protein standards. Concentrations werenormalized to 30 μg, and samples were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein sampleswere transferred to polyvinylidene fluoride (PVDF, Millipore, IPFL00010)membrane at 1.4 amps for 3.5 hours. Membranes were probed overnight at4° C. with antibodies to cleaved Caspase-3 (Cell Signaling, 9661S), CMA1(Gene Tex, GTX72388), and GAPDH (Calbiochem, CB1001). The membranes werevisualized with ECL substrate (GE Healthcare, RPN2132) and film. Westernblot band intensity was quantified as integrated density by densitometryand normalized to the density of loading control.

Any suitable TRPV1 inhibitor or combination of TRPV1 inhibitors may beused in the compositions and methods of the present invention Inhibitorsof TRPV1 family members, as used herein, are substances that reduce(partially, substantially, or completely block) the activity of one ormore members of the TRPV1 family, that is, Trpv1, among others. Thesubstances may be compounds (small molecules of less than about 10 kDa,peptides, nucleic acids, lipids, etc.), complexes of two or morecompounds, and/or mixtures, among others. Furthermore, the substancesmay inhibit TRPV1 family members by any suitable mechanism includingcompetitive, noncompetitive, uncompetitive, mixed inhibition, and/or bychanging a subject's pH, among others. The expression “TRPV1 inhibitor”may refer to a product which, within the scope of sound pharmacologicaljudgment, is potentially or actually pharmaceutically useful as aninhibitor of TRPV1, and includes reference to substances which comprisea pharmaceutically active species and are described, promoted, and/orauthorized as a TRPV1 inhibitor. The strength of inhibition for aselective inhibitor may be described by an inhibitor concentration atwhich inhibition occurs (e.g., an IC₅₀ (inhibitor concentration thatproduces 50% of maximal inhibition) or a K_(i) value (inhibitionconstant or dissociation constant)) relative to different TRPV1 familymembers.

Any suitable TRPV1 inhibitor or combination of inhibitors may be used inthe methods and compositions herein. For example, a subject may betreated with a TRVP1 selective inhibitor and a nonselective TRPV1inhibitor.

-   -   TRPV1 inhibitors include, but are not limited to:

(N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl) Bio Trendtetrahydropyrazine-1(2H)-carboxamide (BCTC) (Switzerland)N-(3-Methoxyphenyl)-4-chlorocinnamide (SB-366791) Neurosci. Lett. 385:137-142 1-lsoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea Eur. J.Pharmacol. (A-425619) 596: 62-69(2E)—N-(2,3-Dihydro-1,4-benzodioxin-6-yl)-3-[4-(1,1- J. Med. Chem.dimethylethyl)phenyl]-2-propenamide (AMG-9810) 50: 3515-3527 (AZD1386)Phase II- AstraZeneca 2-Acetylamino-4-[6′-(4-trifluoromethylphenyl)-pyrimidin-4′-yl-oxy]-benzothiazole (AMG517)N-(2-bromophenyl-N′-[((R)-1-(5-trifluoromethyl- Phase II2-pyridyl)pyrrolidin-3-yl)]urea (SB705498)N-(2-bromophenyl)-N′-{2-[ethyl(3- methylphenyl)amino]ethyl}-urea(SB-452533) ((R)-(5-tert-butyl-2,3-dihydro-1H-inden-1-yl)-3-(1H-indazol-4-yl)-urea (ABT-102)N-(Isoquinolin-5-yl)-N′-[spiro-(cyclobutane-1,2′- Phase II(3′,4′-dihydro-benzopyran-4′-yl))]-urea (GRC-6211)(2R)-4-(3-chloro-2-pyridinyl)-2-methyl-N-[4-(trifluoromethyl)phenyl]-1-piperazinecarboxamide4-(4′-Trifluoromethyl-anilino)-7-(3′-trifluoromethyl-pyridin-2- Phase IIyl)-quinazoline (MK-2295) JYL 1421 Eur. J. Pharmacol. 517: 35-44N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide (Capsazapine)(5R*,8R*,6E,9E)-5,8-Dimethyl-4-methylenetetradeca-6,9- dienoic acid1-(3-Fluorobenzyl)-2-(N-(1,2-dimethyl-1,3-isoindazol-5-yl)-acetamido)-{pyridine-[3,4-b]-pyrrole} (SAR-115740)N-(4-chlorobenzyl)-N′-(1-methyl-1H-indazol-4-yl)urea, AbbottN-(4-tert-butylbenzyl)-N′-(1-methyl-1H-indazol-4-yl)urea, LaboratoriesN-(3-fluoro-4-(trifluoromethyl)benzyl)-N′-(1- (20100249203)methyl-1H-indazol-4-yl)-urea,N-(4-fluoro-3-(trifluoromethyl)-benzyl)-N′-(1-methyl-1H-indazol-4-yl)-urea,N-(3,4-dichlorobenzyl)-N′-(1-methyl-1H-indazol-4-yl)urea,N-(2,4-dichlorobenzyl)-N′-(1-methyl-1H-indazol- 4-yl)urea,N-(4-ethylbenzyl)-N′-(1-methyl-1H-indazol-4-yl)urea,N-(2-chlorobenzyl)-N′-(1-methyl-1H-indazol-4-yl)urea,N-(4-fluorobenzyl)-N′-(1-methyl-1H-indazol-4-yl)urea,N-(2-fluorobenzyl)-N′-(1-methyl-1H-indazol-4-yl)urea,N-[1-(bromophenyl)ethyl-N′-(1-methyl-1H- indazol-4-yl)urea,N-(1-methyl-1H-indazol-4-yl)-N′-{4- [(trifluoromethyl)thio]benzyl}urea.1-(2,3-dichlorophenyl)-3-[2-(N-ethyl-3- methylanilino)ethyl]urea1-[2-(N-ethyl-3-methylanilino)ethyl]-3-naphthalen-1- ylurea1-(4-bromophenyl)-3-[2-(N-ethyl-3- methylanilino)ethyl]urea1-(3-bromophenyl)-3-[2-(N-ethyl-3- methylanilino)ethyl]urea1-(chlorophenyl)-3-[2-(N-ethyl-3- methylanilino)ethyl] urea1-[2-(N-ethyl-3-methylanilino)ethyl]-3-(2- fluorophenyl)urea1-[2-{N-ethyl-3-methylanilino)ethyl]-3-(2- methylphenyl)urea1-[2-(N-ethyl-3-methylanilino)ethyl]-3-phenylurea2-[(2-bromophenyl)carbamoylamino]ethyl-ethylmethyl-(3-methylphenyl)azanium iodide1-(2-bromophenyl)-3-[2-(N-ethyl-3-fluoro-4- methylanilino)ethyl] urea1-(2-bromophenyl)-3-[2-(N-ethyl-3,4- difluoroanilino)ethyl] urea1-(2-bromophenyl)-3-[2-(N-ethyl-3- fluoroanilino)ethyl] urea1-(2-bromophenyl)-3-[2-(N-ethyl-4- methylanilino)ethyl]urea1-(2-bromophenyl)-3-[2-(N-ethyl-2- methylanilino)ethyl]urea1-(2-bromophenyl)-3-[2-(N-ethylanilino)ethyl]ureaN-[2-[(2-bromophenyl)carbamoylamino]ethyl]-N-(3- methylphenyl)acetamide1-[2-{N-benzyl-3-methylanilino)ethyl]-3-(2- bromophenyl)urea1-(2-bromophenyl)-3-[2-(2,3-dimethylanilino)ethyl] urea1-(2-bromophenyl)-3-[2-(3-methylaniIino)ethyl]urea1-(2,5-dichlorophenyl)-3-[2-(N-ethyl-3- methylanilino)ethyl] urea2,6-bis-(4-hydroxy-3-methoxybenzylidene)cyclohexanone (BHMC)4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide4-fluoro-4(pyridin-2-yl)N-[4-trifluoromethylphenyl]piperidine-1-carboxamide4-fluoro-4(pyridine-2-yl)N-[4-trifluoromethylbenzyl]piperidine-1-carboxamide2-{4-fluoro-1-[4-trifluoromethylbenzoyl]piperidin-4-yl}pyridine2-(4-fluoro-1-{[4-trifluoromethylphenyl]acetyl}piperidin-4- yl)pyridine2-(4-fluoro-1-{3-[4-trifluoromethylphenyl]propanoyl}piperidin-4-yl)pyridine 4-fluoro-4-(1-methyl-1H-imidazol-2-yl)-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide4-methoxy-4-pyridin-2-yl-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide4-methoxy-4-pyridin-2-yl-N-[4-trifluoromethylbenzyl]piperidine-1-carboxamide4-fluoro-N-(4-isopropylphenyl)-4-(3-methylpyridin-2-yl)piperidine-1-carboxamide4-fluoro-4-(3-methylpyridin-2-yl)-N-{4-[1,2,2,2-tetrafluoro-1-trifluoromethyl-ethyl]phenyl}piperidine-1-carboxamideN-(4-Tert-butylphenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carboxamide4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-(pentafluoro- lambda(sup6)-sulfanyl)phenyl]piperidine-1-carboxamideN-(4-Butylphenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carboxamide N-(4-Benzylphenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carboxamideN-biphenyl-4-yl-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carboxamide4-fluoro-4-(3-methylpyridin-2-yl)-N-[5-trifluoromethylpyridin-2-yl]piperidine-1-carboxamide 4-(3-chloropyridin-2-yl)-4-fluoro-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide4-fluoro-4-(3-fluoropyridin-2-yl)-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide4-fluoro-4-(3-methoxypyridin-2-yl)-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide 4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-trifluoromethylphenyl]piperidine-1-carbothioamide N′-cyano-4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-trifluoromethylphenyl]piperidine-1-carboximidamide4-fluoro-4-(3-methylpyridin-2-yl)-N′-(1-phenylpiperidin-4-yl)-N-[4-trifluoro-methylphenyl]piperidine-1-carboximidamide4-fluoro-4-phenyl-N-[4-trifluoromethylphenyl]piperidine-1- carboxamide(+/−)-(syn)-4-fluoro-2-methyl-4-(3-methylpyridin-2-yl)-N-[4-trifluoromethyl-phenyl]piperidine-1-carboxamide4-(fluoromethyl)-4-pyridin-2-yl-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide syn- andanti-3-fluoro-3-pyridin-2-yl-N-[4-trifluoromethylphenyl]-8-azabicyclo[3.2-.1]octane-8- carboxamide3-fluoro-3-pyridin-2-yl-N-[4-trifluoromethylphenyl]-8-azabicyclo[3.2.1]octane-8-carboxamide 4-fluoro-4-pyrimidin-2-yl-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide4-fluoro-4-(3-phenylpropyl)-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide2-[4-fluoro-4-(3-methylpyridin-2-yl)piperidin-1-yl]-6-trifluoromethyl-1H-benzimidazole2-(4-fluoro-4-pyridin-2-ylpiperidin-1-yl)-6-(trifluoromethyl)-1H-benzimidazole 4-fluoro-N-[4-trifluoromethylphenyl]-4-[3-trifluoromethylpyridin-2-yl]piperidine-1-carboxamide4-fluoro-N-(4-methylphenyl)-4-(3-methylpyridin-2-yl)piperidine-1-carboxamideN-(4-ethylphenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carboxamide N-(4-chlorophenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carboxamide 4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-trifluoromethoxyphenyl]piperidine-1-carboxamide N-(4-cyanophenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1- carboxamideN-[4-dimethylaminophenyl]-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carboxamide1-(2-(3,3-dimethylbutyl)-4-(trifluoromethyl)benzyl)-3-(1-methyl-1H-indazo-1-4-yl)urea N-acetyl-1-phenylalanyl-1-leucinamideNobilamides A-H (Lin 2011) SB366791 (Gunthrope 2004) TRPV1 antagonists(Messeguer 2006) Capsaicin receptor ligands PCT WO 02/08221

Pharmaceutically acceptable salts forming part of this invention includebase addition salts such as alkali metal salts like Li⁺, Na⁺, and K⁺salts, alkaline earth metal salts like Ca⁺ and Mg⁺ salts, salts oforganic bases such as lysine, arginine, guanidine, diethanolamine,choline and the like, ammonium or substituted ammonium salts. Salts mayinclude acid addition salts which are sulphates, nitrates, phosphates,perchlorates, borates, hydrohalides, acetates, tartrates, maleates,citrates, succinates, palmoates, methanesulphonates, benzoates,salicylates, hydroxynaphthoates, benzenesulfonates, ascorbates,glycerophosphates, ketoglutarates and the like. The termpharmaceutically acceptable solvates includes combinations of solventmolecules with molecules or ions of the solute compound (the inhibitor).Pharmaceutically acceptable solvates may be hydrates or comprising othersolvents of crystallization such as alcohols.

Preferred salts for the list of compounds above are hydrochloride,hydrobromide, sodium, potassium or magnesium.

The present invention provides pharmaceutical compositions containing aTRPV1 inhibitor or mixture of TRPV1 inhibitors. An inhibitor may be inthe form if a pharmaceutically acceptable salt or a pharmaceuticallyacceptable solvate in combination with the usual pharmaceuticallyemployed carriers, diluents and the like.

The pharmaceutical composition may be in the forms normally employed,such as tablets, capsules, powders, syrups, solutions, suspensions andthe like, may contain flavorants, sweeteners etc. in suitable solid orliquid carriers or diluents, or in suitable sterile media to forminjectable solutions or suspensions. Such compositions typically containfrom 1 to 25%, preferably 1 to 15% by weight of active compound, theremainder of the composition being pharmaceutically acceptable carriers,diluents, excipients or solvents.

Suitable pharmaceutically acceptable carriers include solid fillers ordiluents and sterile aqueous or organic solutions. The active compoundwill be present in such pharmaceutical compositions in the amountssufficient to provide the desired dosage in the range as describedabove. Thus, for oral administration, the compounds can be combined witha suitable solid or liquid carrier or diluent to form capsules, tablets,powders, syrups, solutions, suspensions and the like. The pharmaceuticalcompositions, may, if desired, contain additional components such asflavorants, sweeteners, excipients and the like. Pharmaceuticallyacceptable solutions in sesame or peanut oil, aqueous propylene glycoland the like can be used, as well as aqueous solutions of water-solublepharmaceutically-acceptable acid addition salts or alkali or alkalineearth metal salts of the compounds. The injectable solutions prepared inthis manner can then be, administered intravenously, intraperitonally,subcutaneously, or intramuscularly, with intramuscular administrationbeing preferred in humans.

The pharmaceutical compositions of the invention are shown to beeffective by tests in animal models. The pharmaceutical compositions ofthe invention are thus effective for treatment of cardiac hypertrophy ina mammalian subject, including cardiac remodeling, cardiac fibrosis,apoptosis, hypertension, or heart failure. The compositions may also beadministered for prophylactic treatment of cardiac hypertrophy in amammalian subject.

Generally, the effective dose for treating a particular condition in apatient may be readily determined and adjusted by the physician duringtreatment to alleviate the symptoms or indications of the condition ordisease. Generally, a daily dose of active compound (inhibitor) in therange of about 0.01 to 1000 mg/kg of body weight is appropriate foradministration to obtain effective results. The daily dose may beadministered in a single dose or divided into several doses. In somecases, depending upon the individual response, it may be necessary todeviate upwards or downwards from the initially prescribed daily dose.Typical pharmaceutical preparations normally contain from about 0.2 toabout 500 mg of active compound of formula I and/or its pharmaceuticallyactive salts or solvates per dose.

The term “therapeutically effective amount,” “pharmaceutically effectiveamount,” or “effective amount” refers to that amount of a compound ormixture of compounds of Formula I that is sufficient to effecttreatment, as defined below, when administered alone or in combinationwith other therapies to a mammal in need of such treatment. The term“mammal” as used herein is meant to include all mammals, and inparticular humans. Such mammals are also referred to herein as subjectsor patients in need of treatment. The therapeutically effective amountwill vary depending upon the subject and disease condition beingtreated, the weight and age of the subject, the severity of the diseasecondition, the dosing regimen to be followed, timing of administration,the manner of administration and the like, all of which can readily bedetermined by one of ordinary skill in the art.

The term “treatment” or “treating” means any treatment of a disease in amammal, including:

-   -   a) preventing the disease or condition, that is, causing the        clinical symptoms of the disease not to develop;    -   b) inhibiting the disease, that is, slowing or arresting the        development of clinical symptoms; and/or    -   c) relieving the disease, that is, causing the regression of        clinical symptoms.

The invention is explained in detail in the examples given below whichare provided by way of illustration only and therefore should not beconstrued to limit the scope of the invention.

Example 1 Trpv1 Knockout Suppresses Pressure Overload CardiacHypertrophy

Pressure overload cardiac hypertrophy was modeled by transverse aorticconstriction (TAC) in 8 week old male TRPV1 knockout mice (Caterina,2000) and wild type controls. Sham control mice underwent the sameprocedure except for aortic constriction. Baseline pressures wereassessed proximal; and distal, to the TAC banding site, as analyzed byDoppler echo. There was no significant difference between Trpv1^(−/−)and control animals. Transthoracic echocardiography (Echo) was performedusing a high resolution Vevo 770™ Echo system with a 30 MHz transducer(Visual Sonics, Toronto, Canada) in unanethestized mice, in order toassess heart dimensions during pressure overload cardiac hypertrophy, ascompared to sham controls. Mice were sacrificed at 6 weeks post TAC, andhearts were collected for histological sectioning, RNA extraction, andprotein analysis by Western blot and other methods. Gravimetric analysesof cardiac hypertrophy at 6 weeks after TAC, indicate that the heartweight/body weight ratio, as well as the heart weight/tibia length ratioincreases more in the control animals than the Trpv1^(−/−) hearts, ascompared to sham animals. Referring to FIG. 1A the effects of TAC areshown in banded animals on left ventricle (LV) internal diameter,diastolic, from baseline to 6 weeks in C57BU6 WT (n=17), Trpv1^(−/−)(n=15), compared to those effects (FIG. 1B) in sham operated animals(n=9) (middle). In FIG. 1C the effects of TAC are shown on rate of LVIDchange/day in C57BU6 WT (n=17) versus Trpv1^(−/−) (n=15). (P<0.05) WTand Trpv1^(−/−) (p<0.05). In FIG. 1D the effects of TAC on treatedanimals on LV function (fractional shortening) from baseline to 8 weeksin C57BU6

WT (n=17), Trpv1^(−/−) (n=15), and on sham operated animals C57BU6 WT(n=9), Trpv1^(−/−) (n=9). Most notably, in FIG. 1C there is shown asignificant increase in the rate of left ventricle internal diameter, incontrol mice (n=17) as compared to Trpv^(−/−) mice (n=15) (P<0.05). ShamTrpv^(−/−) and control animals did not differ significantly. FIG. 1Dindicates that left ventricular function as measured by percentagefractional shortening

(% FS=([LVDd-LVDs]/[LVDd)×100) appears to be preserved in Trpv1^(−/−)mice from zero to four weeks, as compared to control animals, butdeclines from four to six weeks.

Example 2 Trpv1 Knockout Alters Hypertrophic Markers in PressureOverload Cardiac Hypertrophy

Multiple hypertrophy markers were analyzed from extracted heart lysatesand sections. Overall, major hypertrophic indicators like collagen (FIG.2), atrial natriuretic peptide and TGFβ (FIGS. 3A, 3B) show asignificant reduction in expression in Trpv1^(−/−) mice modeled withpressure overload cardiac hypertrophy for six weeks. In FIG. 2quantification of picrosirius staining in TRPV1 deficient and C57BU6 WTcontrols are shown, performed in Image J, excluding perivascular tissue.Each heart was stained in replicates of 3. Image J was used to analyze 5images from each heart (×3) and determine the pixel count in each fieldas percentage of overall number of pixels for a ratio of red-stainedcollagen/fiber:total tissue area. Images were averaged for each animaland graphed in Prism GraphPad; p<0.001 between TRPV1 banded and C57BU6banded. In FIG. 3A are shown TGF-beta RNA expression in C5781/6 (n=7),and TRPV1−/− (n=4) (p<0.0328). In FIG. 3B are shown ANP expression inC5781/6 (n=15), and TRPV1 (n=14) (p<0.0431). Total RNA was isolated fromhomogenized hearts with Trizol (Molecular Research Center) and furtherpurified with a RNA isolation kit (Mo Bio Laboratories, Inc).Single-stranded eDNA was synthesized from 1 μg of total RNA using a cDNAsynthesis kit (Qiagen). The mRNA levels were quantified by RT-PCR usingSYBR green method.

Example 3 Extracellular Matrix Remodeling

The composition of cardiac tissue changes during the development ofventricular hypertrophy and leads to structural remodeling of themyocardium. One of these changes is related to the disruption of theequilibrium between the synthesis and degradation of collagen, whichresults in an excessive accumulation of collagen type I and III fiberswithin the myocardium. As collagen and other extracellular matrixcomponents accumulate in the interstitial space, myocardial stiffnessincreases and diastolic and systolic dysfunction occurs. Prior dataindicates less interstitial collagen deposition in the Trpvr1^(−/−) micethan control mice, with pressure overload cardiac hypertrophy (Buckley2011). Similar results were obtained by collagen protein assay (Sircol™,Biocolor, Northern Island), and RealTime-PCR. Changes are also seen inthe enzymes responsible for degradation of collagen, the matrixmetalloproteinases (MMPs). (FIGS. 4A, 4B)

The RNA expression changes in sham vs. TAC treated mice shown in FIG. 4Afor MMP2 by RT-PCR (p<0.05), C5781/6 (n=15), and Trpv1^(−/−)(n=14), andin FIG. 4B for MMP 13 by RT-PCR. Generally, suppression of Mmp and Timptranscription is observed more in the Trpv1^(−/−) mice than in controlmice 6 weeks post TAC. However, MMP13 appears upregulated. MMP13 targetscollagen type I, II and III and may serve to protect tissue fromfibrosis. Mast cell chymase (CMA 1) message and protein is expressedless in Trpv1^(−/−) mice than in control mice 8 weeks post TAC (FIG.7D). CMA1 is a chymotryptic serine proteinase that belongs to thepeptidase family S1. It is described as expressed in mast cells butappears to be expressed in other tissues and cell types. It functions inthe degradation of the extracellular matrix and the generation ofvasoactive peptides. In the heart and blood vessels, this protein,rather than angiotensin converting enzyme (ACE), is largely responsiblefor converting angiotensin I to the vasoactive peptide angiotensin II inthe renin-angiotensin system. This system controls blood pressure and isinvolved in the pathogenesis of hypertension, cardiac hypertrophy, andheart failure.

Example 4 Involvement of TRPV1 in the Progression of Cardiac Hypertrophy

Mice lacking functional TRPV1 and control mice with wild-type TRPV1 weremodeled for pressure overload cardiac hypertrophy. Heart dimensions andfunction were measured and compared over time using unanesthestizedtransthoracic echocardiography and hearts were harvested eight weekslater for molecular, biochemical and histological analysis. Heartdimensions and function were better preserved in mice lacking functionalTRPV1. Cellular hypertrophy, markers for hypertrophy, fibrosis andapoptosis were also significantly reduced in these mice, indicatinginvolvement of TRPV1 in the progression of cardiac hypertrophy.

Pressure Overload Model

To test the involvement of TRPV1 in the remodeling associated withcardiac hypertrophy and heart failure, ten-week-old maleB6.129X1-Trpv1tm1Jul/J mice (TRPV1 KO), (Caterina, 2000) and age/sexmatched C57BL/6J (WT) control mice were subjected to acute pressureoverload by transverse aortic constriction (TAC). Sham operated controlmice from both strains underwent an identical surgical procedure exceptfor actual aortic constriction. TRPV1 KO TAC mice and WT TAC mice showedno difference in baseline pressures, assessed immediately distal to theTAC banding site by Doppler echocardiography.

Gravimetric Analysis of the Heart, after Pressure Overload CardiacHypertrophy

This analysis reveals that TAC treated hearts were 28% heavier in WT TACmice than TRPV1 KO TAC mice. When normalized to body weight and tibialength, the heart weight/body weight ratio and the heart weight/tibialength ratio were also significantly greater in WT TAC mice than TRPV1KO TAC mice. (FIGS. 5A and 5B) Mice lacking functional TR PV1 presentpreservation of heart structure and function during pressure overloadcardiac hypertrophy. (▪WT

TR PV1 KO). FIG. 5A is a graph showing heart weight/body weight (HW/BW)and heart weight/tibia length (HW/TL). There is significant differencein HW/BW between WT Sham and TAC mice (p=0.027), WT TAC and TR PV1 KOTAC mice (p=0.019) and TR PV1 KO Sham and TAC mice (p=0.045). FIG. 5Bshows that there is significant difference in HW/TL between WT Sham andTAC mice (p=0.034), WT TAC and TR PV1 KO TAC mice (p=0.03), but notbetween TR PV1 KO Sham and TR PV1 KO TAC mice (p=0.095).

Heart Structure and Function are Maintained During Pressure OverloadCardiac Hypertrophy in Mice Lacking Functional TRPV1

End-diastolic left ventricular internal diameter (LVIDd) was analyzedfor eight weeks following TAC by transthoracic echocardiographicanalysis. In WT TAC mice, LVIDd began to increase at two weeks andplateaued at approximately six weeks. The TRPV1 KO TAC mice showed nochange in LVIDd until six weeks. FIG. 5C shows the analysis of leftventricular internal diameter end-diastolic (LVIDd) from zero to eightweeks in WT (n=6) and TR PV1 KO mice (n=8). The TAC WT control micestart increasing their internal diameter at two weeks, whereas in TAC TRPV1 KO mice there is a delay until six weeks post TAC treatment. Therate of increase in LVIDd is significantly greater in WT TAC mice thanin TRPV1 KO TAC mice (FIG. 5D) between weeks two and six post TAC. FIG.5D shows the rate of change in LVIDd from zero to eight weeks wassignificant (p=0.013) between TAC WT and TAC TR PV1 KO. Heart functionwas analyzed by left ventricular ejection fraction (% EF). Heartfunction declined in WT mice from approximately two to six weeks postTAC treatment, but was preserved in TRPV1 KO TAC mice over the sameperiod of time. FIG. 5E shows a reduction in function starting at twoweeks in TAC WT mice, but TAC TR PV1 KO mice are protected until sixweeks, the percent change in ejection fraction was significantlydifferent at six weeks (p=0.039). The change in ejection fraction at sixweeks is significantly different between WT TAC mice and TRPV1 KO TACmice (FIG. 5F).

Mice Lacking Functional TRPV1 are Protected from Hypertrophy andApoptosis after Modeled Pressure Overload Cardiac Hypertrophy

The degree of cellular hypertrophy was examined by staining of theplasma membranes with fluorescently-labeled wheat germ agglutinin (WGA).Cell sizes were compared by imaging and computer aided measurement ofthe cross-sectional area of cardiomyocytes. This comparison reflects thedegree of cellular hypertrophy between samples. (Shiojima, 2005) Thedata show a significant increase in the cardiomyocyte cross sectionalarea of WT TAC compared to TRPV1 KO TAC mice (FIG. 6A). Measurement ofcardiomyocyte cross sectional area, was significantly different betweenTAC WT mice and TAC TR PV1 KO mice (p=0.025, n=100), 8 weeks post TACtreatment. This shows that, at the cellular level, TRPV1 KO mice developless cardiac hypertrophy than WT mice, in response to modeled pressureoverload cardiac hypertrophy. To further compare the degree ofhypertrophy between TRPV1 and WT mice, additional markers ofhypertrophy, apoptosis and heart failure were assessed. Plasmaconcentrations of the circulating hormone atrial natriuretic peptide(ANP) and the growth factor TGFbeta increase during heart failure andare considered late markers of cardiac hypertrophy. Therefore,expression of ANP and TGFbeta was analyzed by RT-PCR of mRNA isolatedfrom heart tissue. Significantly greater increases were shown in ANP andTGFbeta transcript levels in WT TAC mice than in TRPV1 KO TAC mice. FIG.6B shows that expression levels of atrial natriuretic peptide (ANP) andTGFbeta transcripts were significantly greater in TAC WT mice than inTRPV1 KO mice (p=0.037, p=0.007) relative to control mice. Western blotanalysis confirmed that there was a significant increase (FIG. 6C) inANP protein expression in TAC WT mice compared to TAC TRPV1 KO mice.These data show that protection from the stress and or signaling systemsassociated with the hypertrophic transcriptional responses is observedin the TRPV1 KO mice. The degree of cellular apoptosis by measurement ofcleaved caspase-3 protein in heart tissue lysates from TAC and shamtreated WT and TRPV1 KO mice were assessed. Analysis of western blotdensitometry of heart tissue lysates showed significantly less caspase-3cleavage in TRPV1 KO TAC mice than in WT TAC mice. As expected, WT shamand TRPV KO sham mice showed no apparent caspase-3 cleavage. (FIG. 6D)These results show that TAC-induced cardiac apoptosis is reduced inTRPV1 KO mice. There is protection from the stress and or signalingassociated with cardiac hypertrophy in the TRPV1 KO mice.

Mice Lacking Functional TRPV1 Show Reduced Fibrosis, Tissue Remodelingand Inflammatory Markers after Modeled Pressure Overload CardiacHypertrophy

During the development of ventricular hypertrophy, the composition ofcardiac tissue changes, leading to structural remodeling of themyocardium. For example, the disruption of the equilibrium between thesynthesis and degradation of collagen results in an excessiveaccumulation of collagen type I and III fibers within the myocardium. Ascollagen and other extracellular matrix components accumulate in theinterstitial space, myocardial stiffness increases, and diastolic andsystolic dysfunction occurs. Collagen III levels were analyzed by RT-PCRand total collagen by histological staining, in heart tissue from Shamand TAC, WT and TRPV1 KO mice. It was shown that collagen III transcriptlevels (FIG. 7A), and interstitial collagen deposition (FIG. 7B) werereduced in hearts isolated from TRPV1 KO TAC mice compared to WT TACmice. Mice lacking functional TR PV1 present with less interstitialfibrosis and tissue remodeling enzymes than WT control mice, eight weekspost TAC treatment. (▪WT

TR PV1 KO). FIG. 7A shows an increase in the expression of Collagen IIItranscript was significantly greater in TAC WT mice than in TAC TRPV1 KOmice (p=0.037, 0.007). In FIG. 7B mice lacking functional TR PV1 presentwith less interstitial fibrosis and tissue remodeling enzymes than WTcontrol mice, eight weeks post TAC treatment. (▪WT

TR PV1 KO). There was a significant increase in MMP2 transcripts inhearts from WT TAC mice compared to hearts from TRPV1 TAC mice. FIG. 7Cshows that increases in the expression matrix metalloproteinase-2 (MMP-2) transcript was significantly less in TAC treated TR PV1 KO micethan TAC treated WT mice (p=0.049). There was also significantly lessexpression of Chymase (CMA1) transcript (p=0.049). Mast cell chymase,CMA1, is a chymotryptic serine proteinase that belongs to the peptidasefamily 51. It functions in the degradation of the extracellular matrixand in the generation of vasoactive peptides. In the heart and bloodvessels, it is CMA1, rather than angiotensin converting enzyme (ACE),that is largely responsible for converting angiotensin I to thevasoactive peptide angiotensin II. The data in FIGS. 7D and 7E show thatCMA1 transcripts and protein are expressed at significantly higherlevels in hearts from WT TAC mice than TRPV1 KO TAC mice. There wassignificantly less expression of Chymase (CMA1) transcript (p=0.049),and Chymase protein (p=0.0218) in isolated heart tissue from TAC TR PV1KO mice than TAC WT mice (CMA1 integrated density was normalized toGAPDH loading control). The data show that the functional knockout ofTRPV1 in mice allows for the preservation of heart structure and heartfunction under modeled pressure overload. Concomitant with thisprotection is the down-regulation of multiple protein andtranscriptional markers associated with initiation and the progressionof hypertrophy, apoptosis, fibrosis, and heart failure. This data showthat TRPV1 has a role as either an initiating stressor, or an upstreamsignaling transducer of the hypertrophic transcriptional response in theheart.

Example 5

Mice Treated with the TRPV1 Antagonist BCTC Present Preservation ofHeart Mass, Structure and Function During Pressure Overload CardiacHypertrophy

The following tests show that treatment by continuous administrationusing osmotic pumps with the TRPV1 antagonist, BCTC, in WT mice exposedto TAC confirms the findings from tests of the prior Examples.

Osmotic Pump Installation.

Long term (up to 42 day) infusion of drugs can be accomplished byinsertion of osmotic pumps without the need for repeated injection. Miceare placed under a low plane of anesthesia with an injection ofKetamine/Xylazine anesthetic (50 mg/10 mg·Kg) intraperitoneally (IP) 10minutes prior to surgery. A small area between shoulder blades is shavedand sterilized with Povidine swab. A small incision is made in this areaand blunt dissected below skin to allow placement of an Alzet osmoticpump (model 2006), previously loaded with the drug of choice under theskin between the shoulder blades where it is inaccessible to the mouse.Several stitches are applied to close the incision. The mouse is placedin regular housing on a warming mat until completely conscious, afterwhich mice are then returned to regular housing room.

The mice were subjected to pressure overload induced cardiac hypertrophyby TAC while administered 4 mg/kg of BCTC (in 20% wt/vol2-Hydroxypropyl)-β-cyclodextrin/PBS) throughout the entire experimentusing osmotic pumps (Alzet, Model 2006) pumping continuously at a rateof 0.15 ul/hr. The test was limited to ˜42 days (max) by the function ofthe pumps, as such the experiment was halted at 36 days post TAC, aspumps are installed previous to the TAC to allow recovery before the TACsurgery. Analysis of heart weights 36 days post TAC revealed that theheart weight/body weight ratio was significantly greater in vehicletreated WT mice than drug treated mice (p=0.035) (FIG. 8A).Echocardiographic assessment of mice every 9 days for the duration ofthe study showed that Vehicle and BCTC treated sham mice (Vehicle Sham(n=2) and BCTC sham mice (n=2)) show no difference in their leftventricular internal diameter (LVID,d) (FIG. 8B). However, BCTC treatedTAC mice (n=8) have a significantly smaller LVIDd than the vehicletreated TAC mice (n=7) (FIG. 8C) from zero to thirty six days,indicating that the TAC Vehicle control mice start increasing theirinternal diameter at 9 days, whereas in TAC BCTC treated mice, thediameter is maintained for the duration of the test. The LVIDd issignificantly different after 18 days (p<0.001). This protection fromdilation of the left ventricle translates to a protection in thefunction of the heart as measured by ejection fraction (% EF, p<0.05)and fractional shortening (% FS). Both the % EF and % FS (p<0.01) ofvehicle-treated TAC mice declined steadily over the course of the studyand was significantly diminished at 36 days post TAC compared todrug-treated mice (FIGS. 8D, 8E).

Example 6

Mice Treated with the TRPV1 Antagonist BCTC Present Histologically withLess Hypertrophy and Fibrosis than Vehicle Control Mice, Thirty Six DaysPost TAC Treatment

Histological analysis of the heart 36 days post TAC shows that BCTC canprotect the heart from cellular hypertrophy, and the deposition ofinterstitial fibrosis. From stains of plasma membranes (Wheat germagglutinin-Alexa488) in heart tissue sections it is shown that BCTCtreated TAC mice, described in Example 5, have smaller cardiac myocytesand less hypertrophy than vehicle treated TAC control mice. Measurementof cardiomyocyte cross sectional area shows significantly smallermyocytes in BCTC treated TAC mice than vehicle treated TAC control mice(p=<0.01, n=100) 36 days post TAC treatment (FIG. 9A), indicating thatBCTC can protect the heart from hypertrophy at the cellular level. Lesshistological staining with Picrosirius red which can indicate areas ofinterstitial collagen deposition in isolated heart tissue sections fromBCTC treated TAC mice than vehicle treated control mice indicates lessinterstitial collagen deposition. Analysis of collagen staining byImageJ (NIH) was used to determine the area of collagen staining as apercentage of tissue area. The analysis (FIG. 9B) indicates that thereis significantly less interstitial collagen in BCTC treated TAC micethan vehicle treated TAC control mice (p=0.05). This shows that BCTC canprotect the heart from fibrosis during pressure overload cardiachypertrophy.

CITATIONS

-   Lygate, C. 2006. Surgical models of hypertrophy and heart failure:    Myocardial infarction and transverse aortic constriction. Drug    Discovery Today: Disease Models 3:283-290.-   Patten, R. D., and M. R. Hall-Porter. 2009. Small animal models of    heart failure: development of novel therapies, past and present.    Circ Heart Fail 2:138-144.-   Rockman, H. A., S. Ono, R. S. Ross, L. R. Jones, M. Karimi, V.    Bhargava, J. Ross, Jr., and K. R. Chien. 1994. Molecular and    physiological alterations in murine ventricular dysfunction. Proc    Natl Acad Sci USA 91:2694-2698.-   Rockman, H. A., R. S. Ross, A. N. Harris, K. U. Knowlton, M. E.    Steinhelper, L. J. Field, J. Ross, Jr., and K. R. Chien. 1991.    Segregation of atrial-specific and inducible expression of an atrial    natriuretic factor transgene in an in vivo murine model of cardiac    hypertrophy. Proc Natl Acad Sci USA 88:8277-8281.-   Caterina, M. J., et al. 2000. Impaired nociception and pain    sensation in mice lacking the capsaicin receptor. Science    288:306-313.-   Buckley, C. L., and Stokes, A. J. 2011. Mice lacking functional    TRPV1 are protected from pressure overload cardiac hypertrophy.    Channels 5:4, 1-8.-   Shiojima, I., Sato, K., Izumiya, Y., Schiekofer, S., Ito, M., Liao,    R., et al. 2005. Disruption of coordinated cardiac hypertrophy and    angiogenesis contributes to the transition to heart failure. J.    Clin. Invest. 115:2108-18.-   Gunthrope, M. J., Rami, H. K., et al. 2004. Discovery of novel    6,6-heterocycles as transient receptor potential vanilloid (TRPV1)    antagonists. Neuropharmacol. 46(1):133-49.-   Lin, Z., Reilly, C. A., et al. 2011. Nobilamides A-H, long-acting    transient receptor potential vanilloid-1 (TRPV1) antagonists from    mollusk-associated bacteria. J. Med. Chem. 54(11):3746-55.-   Messeguer, A., Planells-Cases, R., et al. 2006. Physiology and    pharmacology of the vanilloid receptor. Curr. Neuropharmacol.    4(1):1-15.

1. A method of treating cardiac hypertrophy in a mammalian subjectcomprising administering to the subject an anti-hypertrophic effectiveamount of an ion channel TRPV1 inhibitor.
 2. A method of prophylactictreatment for cardiac hypertrophy in a mammalian subject comprisingadministering to the subject an anti-hypertrophic effective amount of anion channel TRPV1 inhibitor.
 3. The method according to claim 1 whereina symptom of cardiac hypertrophy in said subject comprises cardiacremodeling.
 4. The method according to claim 1 wherein a symptom ofcardiac hypertrophy in said subject comprises cardiac fibrosis.
 5. Themethod according to claim 1 wherein a symptom of cardiac hypertrophy insaid subject comprises hypertension.
 6. The method according to claim 1wherein a symptom of cardiac hypertrophy in said subject comprises heartfailure.
 7. The method according to claim 1 wherein a symptom of cardiachypertrophy in said subject comprises apoptosis.
 8. The method accordingto claim 1 or 2 wherein inhibitor is selected from the group consistingof(N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)-tetrahydropyrazine-1(2H)-carboxamide N-(3-Methoxyphenyl)-4-chlorocinnamide1-lsoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea(2E)-N-(2,3-Dihydro-1,4-benzodioxin-6-yl)-3-[4-(1,1-dimethylethyl)phenyl]-2-propenamide2-Acetylamino-4-[6′-(4-trifluoromethylphenyl)-pyrimidin-4′-yl-oxy]-benzothiazoleN-(2-bromophenyl-N′-[((R)-1-(5-trifluoromethyl-2-pyridyl)pyrrolidin-3-yl)]ureaN-(2-bromophenyl)-N′-{2-[ethyl(3-methylphenyl)amino]ethyl}urea((R)-(5-tert-butyl-2,3-dihydro-1H-inden-1-yl)-3-(1H-indazol-4-yl)-ureaN-(Isoquinolin-5-yl)-N′-[spiro-(cyclobutane-1,2′-(3′,4′-dihydro-benzopyran-4′-yl))]urea(2R)-4-(3-chloro-2-pyridinyl)-2-methyl-N-[4-(trifluoromethyl)phenyl]-1-piperazinecarboxamide4-(4′-Trifluoromethyl-anilino)-7-(3′-trifluoromethyl-pyridin-2-yl)-quinazolineN-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide(5R*,8R*,6E,9E)-5,8-Dimethyl-4-methylenetetradeca-6,9-dienoic acid1-(3-Fluorobenzyl)-2-(N-(1,2-dimethyl-1,3-isoindazol-5-yl)-acetamido)-{pyridine-[3,4-b]-pyrrole} N-(4-chlorobenzyl)-N′-(1-methyl-1H-indazol-4-yl)urea,N-(4-tert-butylbenzyl)-N′-(1-methyl-1H-indazol-4-yl)urea,N-(3-fluoro-4-(trifluoromethyl)benzyl)-N′-(1-methyl-1H-indazol-4-yl)-urea,N-(4-fluoro-3-(trifluoromethyl)benzyl)-N′-(1-methyl-1H-indazol-4-yl)-urea,N-(3,4-dichlorobenzyl)-N′-(1-methyl-1H-indazol-4-yl)urea,N-(2,4-dichlorobenzyl)-N′-(1-methyl-1H-indazol-4-yl)urea,N-(4-ethylbenzyl)-N′-(1-methyl-1H-indazol-4-yl)urea,N-(2-chlorobenzyl)-N′-(1-methyl-1H-indazol-4-yl)urea,N-(4-fluorobenzyl)-N′-(1-methyl-1H-indazol-4-yl)urea,N-(2-fluorobenzyl)-N′-(1-methyl-1H-indazol-4-yl)urea,N-[1-(bromophenyl)ethyl-N′-(1-methyl-1H-Indazol-4-yl)urea,N-(1-methyl-1H-indazol-4-yl)-N′-{4-[(trifluoromethyl)thio]benzyl}urea.1-(2,3-dichlorophenyl)-3-[2-(N-ethyl-3-methylanilino)ethyl]urea1-[2-(N-ethyl-3-methylanilino)ethyl]-3-naphthalen-1-ylurea1-(4-bromophenyl)-3-[2-(N-ethyl-3-methylanilino)ethyl]urea1-(3-bromophenyl)-3-[2-(N-ethyl-3-methylanilino)ethyl]urea1-(chlorophenyl)-3-[2-(N-ethyl-3-methylanilino)ethyl] urea1-[2-(N-ethyl-3-methylanilino)ethyl]-3-(2-fluorophenyl)urea1-[2-{N-ethyl-3-methylanilino)ethyl]-3-(2-methylphenyl)urea1-[2-(N-ethyl-3-methylanilino)ethyl]-3-phenylurea2-[(2-bromophenyl)carbamoylamino]ethyl-ethylmethyl-(3-methylphenyl)azaniumiodide1-(2-bromophenyl)-3-[2-(N-ethyl-3-fluoro-4-methylanilino)ethyl]urea1-(2-bromophenyl)-3-[2-(N-ethyl-3,4-difluoroanilino)ethyl]urea1-(2-bromophenyl)-3-[2-(N-ethyl-3-fluoroanilino)ethyl]urea1-(2-bromophenyl)-3-[2-(N-ethyl-4-methylanilino)ethyl]urea1-(2-bromophenyl)-3-[2-(N-ethyl-2-methylanilino)ethyl]urea1-(2-bromophenyl)-3-[2-(N-ethylanilino)ethyl]ureaN-[2-[(2-bromophenyl)carbamoylamino]ethyl]-N-(3-methylphenyl)acetamide1-[2-{N-benzyl-3-methylanilino)ethyl]-3-(2-bromophenyl)urea1-(2-bromophenyl)-3-[2-(2,3-dimethylanilino)ethyl]urea1-(2-bromophenyl)-3-[2-(3-methylaniIino)ethyl]urea1-(2,5-dichlorophenyl)-3-[2-(N-ethyl-3-methylanilino)ethyl]urea4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide4-fluoro-4(pyridin-2-yl)N-[4-trifluoromethylphenyl]piperidine-1-carboxamide4-fluoro-4(pyridine-2-yl)N-[4-trifluoromethylbenzyl]piperidine-1-carboxamide2-{4-fluoro-1-[4-trifluoromethylbenzoyl]piperidin-4-yl}pyridine2-(4-fluoro-1-{[4-trifluoromethylphenyl]acetyl}piperidin-4-yl)pyridine2-(4-fluoro-1-{3-[4-trifluoromethylphenyl]propanoyl}piperidin-4-yl)pyridine4-fluoro-4-(1-methyl-1H-imidazol-2-yl)-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide4-methoxy-4-pyridin-2-yl-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide4-methoxy-4-pyridin-2-yl-N-[4-trifluoromethylbenzyl]piperidine-1-carboxamide4-fluoro-N-(4-isopropylphenyl)-4-(3-methylpyridin-2-yl)piperidine-1-carboxamide4-fluoro-4-(3-methylpyridin-2-yl)-N-{4-[1,2,2,2-tetrafluoro-1-trifluoromethyl-ethyl]phenyl}piperidine-1-carboxamideN-(4-Tert-butylphenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carboxamide4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-(pentafluoro-lambda(sup 6)-sulfanyl)phenyl]piperidine-1-carboxamideN-(4-Butylphenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carboxamideN-(4-Benzylphenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carboxamideN-biphenyl-4-yl-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carboxamide4-fluoro-4-(3-methylpyridin-2-yl)-N-[5-trifluoromethylpyridin-2-yl]piperidine-1-carboxamide4-(3-chloropyridin-2-yl)-4-fluoro-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide4-fluoro-4-(3-fluoropyridin-2-yl)-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide4-fluoro-4-(3-methoxypyridin-2-yl)-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide 4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-trifluoromethylphenyl]piperidine-1-carbothioamideN′-cyano-4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-trifluoromethylphenyl]piperidine-1-carboximidamide4-fluoro-4-(3-methylpyridin-2-yl)-N′-(1-phenylpiperidin-4-yl)-N-[4-trifluoro-methylphenyl]piperidine-1-carboximidamide4-fluoro-4-phenyl-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide(+/−)-(syn)-4-fluoro-2-methyl-4-(3-methylpyridin-2-yl)-N-[4-trifluoromethyl-phenyl]piperidine-1-carboxamide4-(fluoromethyl)-4-pyridin-2-yl-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide syn- andanti-3-fluoro-3-pyridin-2-yl-N-[4-trifluoromethylphenyl]-8-azabicyclo[3.2-.1]octane-8-carboxamide3-fluoro-3-pyridin-2-yl-N-[4-trifluoromethylphenyl]-8-azabicyclo[3.2.1]octane-8-carboxamide4-fluoro-4-pyrimidin-2-yl-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide4-fluoro-4-(3-phenylpropyl)-N-[4-trifluoromethylphenyl]piperidine-1-carboxamide2-[4-fluoro-4-(3-methylpyridin-2-yl)piperidin-1-yl]-6-trifluoromethyl-1H-benzimidazole2-(4-fluoro-4-pyridin-2-ylpiperidin-1-yl)-6-(trifluoromethyl)-1H-benzimidazole4-fluoro-N-[4-trifluoromethylphenyl]-4-[3-trifluoromethylpyridin-2-yl]piperidine-1-carboxamide4-fluoro-N-(4-methylphenyl)-4-(3-methylpyridin-2-yl)piperidine-1-carboxamideN-(4-ethylphenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carboxamideN-(4-chlorophenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carboxamide4-fluoro-4-(3-methylpyridin-2-yl)-N-[4-trifluoromethoxyphenyl]piperidine-1-carboxamideN-(4-cyanophenyl)-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carboxamideN-[4-dimethylaminophenyl]-4-fluoro-4-(3-methylpyridin-2-yl)piperidine-1-carboxamide1-(2-(3,3-dimethylbutyl)-4-(trifluoromethyl)benzyl)-3-(1-methyl-1H-indazo-l-4-yl)urea N-acetyl-1-phenylalanyl-1-leucinamide and pharmaceuticallyacceptable salts thereof.


9. The method according to claim 7 wherein the inhibitor comprises(N-(4-t-butyl-phenyl)-4-(3-chloropyridin-2-yl)-tetrahydropyrazine-1(2H)-carboxamide or a pharmaceutically acceptable salt thereof.
 10. Themethod according to claim 1 or 2 wherein the inhibitor comprises(N-(4-t-butyl-phenyl)-4-(3-chloropyridin-2-yl)-tetrahydropyrazine-1(2H)-carboxamide or a pharmaceutically acceptable salt thereof.
 11. Apharmaceutical composition comprising a pharmaceutically effectiveamount of an inhibitor or mixture of inhibitors according to claim 1 anda pharmaceutically acceptable carrier sufficient to alleviateprogression of cardiac hypertrophy in said subject.
 12. A pharmaceuticalcomposition comprising a pharmaceutically effective amount of aninhibitor or mixture of inhibitors according to claim 2 and apharmaceutically acceptable carrier sufficient to inhibit onset ofcardiac hypertrophy in said subject.